Regulatory T Cells in γ Irradiation-Induced Immune Suppression

Sublethal total body γ irradiation (TBI) of mammals causes generalized immunosuppression, in part by induction of lymphocyte apoptosis. Here, we provide evidence that a part of this immune suppression may be attributable to dysfunction of immune regulation. We investigated the effects of sublethal TBI on T cell memory responses to gain insight into the potential for loss of vaccine immunity following such exposure. We show that in mice primed to an MHC class I alloantigen, the accelerated graft rejection T memory response is specifically lost several weeks following TBI, whereas identically treated naïve mice at the same time point had completely recovered normal rejection kinetics. Depletion in vivo with anti-CD4 or anti-CD25 showed that the mechanism involved cells consistent with a regulatory T cell (T reg) phenotype. The loss of the T memory response following TBI was associated with a relative increase of CD4+CD25+ Foxp3+ expressing T regs, as compared to the CD8+ T effector cells requisite for skin graft rejection. The radiation-induced T memory suppression was shown to be antigen-specific in that a third party ipsilateral graft rejected with normal kinetics. Remarkably, following the eventual rejection of the first MHC class I disparate skin graft, the suppressive environment was maintained, with markedly prolonged survival of a second identical allograft. These findings have potential importance as regards the immunologic status of T memory responses in victims of ionizing radiation exposure and apoptosis-inducing therapies

Rejection of allogeneic D^d skin grafts by naïve and D^d primed FVB (H-2^q) mice.

<p>In vivo depletion of CD4 and/or CD8 T cells. On days −6, −5, −1 and +5, GK1.5 (150 µg) mAb and the 53.6.7 (200 µg) mAb were injected IP into adult thymectomized FVB/N mice.</p>*<p>Significant difference from control.</p

Allograft survival is prolonged in alloantigen primed, but not naïve mice, several weeks following γ-irradiation.

<p>A. Groups of 6 FVB mice were immunized IP with 2×10⁷ 3604 (D^d) splenocytes, and 3 wks later given a 550 cGy dose of total body γ radiation (TBI). Mice were engrafted with 3604 tail skin grafts at either 1 or 4 wks following irradiation. B. Radiation induced T memory suppression (RITMS) develops between 2 and 3 weeks following γ-irradiation of immunized mice. Experiment designed as in A, except that separate groups of mice received allogeneic tail skin grafts at 1 or 2 or 3 or 4 weeks post-irradiation (1 wk vs. 3 wks: p<0.01, 2 wks vs. 3 wks. p<0.05, 1 or 2 wks vs. 4 wks, p<0.01, 1 vs. 2 wks, p<0.05). C. Suppression persists after eventual allograft rejection in RITMS mice. Mice from the experiment described in Fig. 1B were given a second identical ipsilateral allograft after the initial graft had rejected (77 d after the initial engraftment). Time to graft rejection is shown for individual mice. One mouse identified as “C” had 30% of the original allograft remaining. This original graft rejected after 149 days. The experiment was terminated after 100 d of observation of the second allograft.</p

Isoform-Specific Expression and Feedback Regulation of E Protein TCF4 Control Dendritic Cell Lineage Specification

The cell fate decision between interferon-producing plasmacytoid DC (pDC) and antigen-presenting classical DC (cDC) is controlled by the E protein transcription factor TCF4 (E2-2). We report that TCF4 comprises two transcriptional isoforms, both of which are required for optimal pDC development in vitro. The long Tcf4 isoform is expressed specifically in pDCs, and its deletion in mice impaired pDCs development and led to the expansion of non-canonical CD8⁺ cDCs. The expression of Tcf4 commenced in progenitors and was further upregulated in pDCs, correlating with stage-specific activity of multiple enhancer elements. A conserved enhancer downstream of Tcf4 was required for its upregulation during pDC differentiation, revealing a positive feedback loop. The expression of Tcf4 and the resulting pDC differentiation were selectively sensitive to the inhibition of enhancer-binding BET protein activity. Thus, lineage-specifying function of E proteins is facilitated by lineage-specific isoform expression and by BET-dependent feedback regulation through distal regulatory elements

Increased proportion of T regs in cell cycle.

<p>Groups of 5 FVB/N mice were primed IP with 2×10⁷ 3604 or FVB/N splenocytes. Four weeks later the mice were exposed to 550 cGy TBI and 11 days later spleens were harvested. (A) Representative plots of splenocytes from unirradiated and irradiated mice stained for expression of CD4 or CD8 and Foxp3 and Ki-67. (B) The percentage of Ki-67+ cells among CD4+Foxp3+ and CD4+Foxp3<b>−</b> cell populations, showing naïve and D^d-primed mice with and without irradiation. For each treatment group, a significantly greater proportion of the CD4+Foxp3+ cells express Ki-67 (p<0.001) than do the CD4+Foxp3<b>−</b> cell population.</p

Expression of Foxp3, CD8 and skin-homing chemokine receptor genes in allogeneic skin grafts and lymph nodes.

<p>D^d primed (<b>P</b>) or unprimed (naïve, <b>N</b>) mice were treated with 550 cGy of γ irradiation (<b>I</b>) and subsequently received 3604 tail skin grafts (<b>G</b>) 21 days after irradiation. Allografts and LN were harvested at 16 days post-engraftment, when (data not shown) primed and unirradiated primed mice had rejected their allografts. Fold increase and ratio of fold increase were compared for Foxp3 and CD8 mRNA for skin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6A</a>), and lymph nodes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6B</a>), and for chemokine receptor expression in allografted skin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6C</a>) by real-time PCR (n = 4−6/group) relative to control untouched skin (<b>C</b>). Statistical significance between groups is indicated in each graph and as assessed by One Way ANOVA and Tukey’s multiple comparison test.</p

Specificity of RITMS.

<p>Groups of 10 mice were primed I.P. with 2×10⁷ syngeneic (FVB), multiple minor allogeneic (DBA/1), or MHC-D^d allogeneic (3604) spleen cells. γ radiation (550 cGy) was given 5 wks later, and mice were engrafted ipsilaterally with both DBA/1 and D^d disparate skin after an additional 6 wks.</p

In vivo depletion with anti-CD4 or anti-CD25 ablates RITMS.

<p>One day after (A,B), or 2 wk before (C,D), 550 cGy TBI, groups of 6 D^d primed (2×10⁷ 3604 spleen cells IP 3 wks prior to TBI) or naïve (equal vol. PBS IP) FVB mice were given a single IP injection of 80 µg of anti-CD4 (GK1.5) or anti-CD25 (PC61) or control rat IgG. Mice in the “untreated” group received no irradiation or antibody treatment. All groups were engrafted with 3604 (D^d) tail skin 4 weeks after irradiation. At 10 d after mAb treatment CD25 and CD4 depletion were >90%. Similar results were obtained in two analogous depletion experiments, one of which used the rat IgG2a GL113 as an isotype control for PC61.</p